PURPOSE: This study investigated the effect of beta-alanine supplementation on short-duration sprints and final 4-km simulated uphill cycling time-trial performance during a comprehensive and novel exercise protocol representative of the demands of road-race cycling, and determined if changes were related to increases in muscle carnosine content. METHODS: Seventeen cyclists (age 38±9 y, height 1.76±0.07 m, body mass 71.4±8.8, VO2max 52.4±8.3 ml·kg-1·min-1) participated in this placebo-controlled, double-blind study. Cyclists undertook a prolonged intermittent cycling protocol lasting 125 minutes, with 6 x 10 s sprints every 20 minutes, finishing with a 4-km time-trial at 5% simulated incline. Participants completed two familiarization and two main sessions pre-supplementation, and one post-supplementation session following 28 days of 6.4 g·day-1 of beta-alanine (N=11) or placebo (N=6; maltodextrin). Muscle biopsies were obtained pre- and post-supplementation for subsequent analysis of muscle carnosine content. RESULTS: There was no group (P=0.69), time (P=0.50) or group x time interaction (P=0.26) on time-to-complete the 4-km time-trial. Time-to-completion did not change from pre- to post- supplementation for BA (-19.2±45.6 s, P=0.43) or PL (+2.8±31.6 s, P=0.99). Beta-alanine did not influence blood lactate values or ratings of perceived exertion during the prolonged cycling test. Beta-alanine supplementation increased muscle carnosine content from pre- to post-supplementation (+9.4±4.0 mmol·kg-1 dm; P<0.0001) but was not related to performance changes. CONCLUSIONS: Chronic beta-alanine supplementation increased muscle carnosine content, however, this did not improve short-duration sprint performance throughout simulated road race cycling, nor performance of a 4-km uphill time-trial conducted at the end of this cycling test. Beta-alanine supplementation may not benefit this type of road cycling performance. [ABSTRACT FROM AUTHOR]